【作者】 张美玉；

【导师】 李连达；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2010， 博士

【摘要】 鱼腥草注射剂作为中药注射剂临床严重不良反应发生的代表药,其再研究和再评价工作具有非常重要的现实意义。对鱼腥草注射剂开展安全性再评价工作,必须从制剂工艺、质量标准、药理毒理、临床使用及药物经济学等方面进行全面的科学评估。本课题组对鱼腥草注射剂临床不良反应的原因进行系统分析研究,明确鱼腥草注射剂中的主要致敏物质,完善生产质量标准,改进鱼腥草注射剂制备工艺,从而提高了注射剂的安全性、有效性和稳定性,为其恢复生产和临床应用提供科学依据。本论文主要包括以下四个内容：1文献综述本论文文献综述部分系统论述了三个方面的内容：①鱼腥草及其制剂研究进展；②鱼腥草注射剂临床不良反应原因分析和对策；③注射用辅料吐温80和羟丙基-β-环糊精研究进展。以上这些论述为论文的整体研究奠定了理论基础。2鱼腥草注射液原、新制剂及其辅料的致敏性评价实验研究本研究分别采用小鼠、Beagle犬和食蟹猴对鱼腥草注射液原、新制剂及其辅料进行致敏性评价研究。类过敏实验以单次静脉恒速注射给药,观察给药后动物的行为变化和血中组胺含量。过敏实验为三次隔日静脉注射给药,此为致敏阶段,在首次给药后14天药量加倍激发给药。同样观察动物的行为学变化和血浆中组胺、IgE的含量,综合判定结果。结果表明类过敏反应以Beagle犬为最佳实验动物,以行为异常及组胺升高为主要判定指标,IgE作为辅助指标,结果准确可靠。食蟹猴在同样条件下不如上述动物敏感。小鼠行为表现更不典型,血液指标的检测误差较大,实验结果仅供参考。实验表明吐温80是诱发类过敏反应的代表性物质。羟丙基-β-环糊精类过敏和过敏反应结果均为阴性。以羟丙基-β-环糊精为增溶剂的鱼腥草注射液新制剂致敏性评价结果为阴性。3鱼腥草注射液新、原制剂比较药理学实验研究抗病毒实验采用流感病毒FM1和PR8病毒株体内感染小鼠肺炎模型,体外CPE法和神经氨酸酶荧光测定法、病毒载量检测和小鼠死亡保护实验方法,结果显示鱼腥草注射液新制剂对流感病毒感染引起的小鼠肺部炎症均有明显治疗和预防作用,可改善小鼠肺炎症状,降低肺指数,病毒载量,降低死亡率,延长小鼠存活时间；但其抗病毒作用并不是通过直接抑制或杀死病毒,对神经氨酸酶活力也不具有抑制作用。新制剂对二甲苯诱发小鼠耳肿胀有明显抑制作用,能降低醋酸导致的小鼠腹腔毛细血管通透性；同时,还能降低干酵母致大鼠体温升高,对脂多糖导致的家兔体温升高有明显的抑制作用。鱼腥草注射液抗炎、解热作用可能是通过抑制炎症因子TNF-α、IL-1β、IL-8和PGE2释放而发挥作用。鱼腥草注射液新、原制剂抗病毒、抗炎解热作用相当。4鱼腥草注射剂中吐温80的定性定量测定方法建立本研究采用硫氰酸钴铵显色法、薄层层析法和碘化铋钾法三种方法进行吐温80的定性鉴定,实验结果一致。吐温80的定量检测方法能准确的检测中药注射剂中吐温80含量。吐温80的定性定量方法的建立在实验室条件下可以随机抽取不同厂家、不同批次样品进行吐温80定性定量测定。本研究主要结论如下：1中药注射剂诱发过敏和类过敏反应的异同比较：中药注射剂导致临床严重不良反应70%以上为类过敏反应,与IgE介导的经典抗原抗体反应不同。主要特征为：首次注射药物即可出现严重不良反应及过敏性休克,与过敏反应症候群相似。属于非抗原抗体反应,IgE、IgG未见明显规律性变化,但体内组胺明显升高。其致敏物质为抗原或半抗原物质及某些可以为诱发组胺释放的多种物质。存在明显的量效关系,大剂量、高浓度可诱发严重不良反应。多种注射剂联合应用可提高发生率及严重性。类过敏反应以Beagle犬为最佳实验动物,以行为异常及组胺升高为主要判定指标,IgE作为辅助指标,结果准确可靠。因此,推荐采用Beagle犬致敏性评价方法作为中药注射剂出厂前的常规安全性检测方法。2鱼腥草注射剂中主要致敏物质为增溶剂吐温80,鱼腥草双蒸馏液无明显的毒副作用。中药注射剂引发不良反应的致敏原除了考虑药物本身因素,还有制剂外加辅料和制备、储存过程中内、外源性污染物,这为寻找和解决中药注射剂不良反应诱发因素提供了思路和方法。3吐温80国内外应用广泛。中药注射剂134品种中吐温80添加情况很不确定。如果这些品种全部换掉将事关几十个注射剂产品,数百家生产企业及几十万产业工人的生存,对社会稳定和经济影响是必须慎重考虑的问题。通过我们研究发现吐温80引起的类过敏反应存在着明显的量效关系。因此,优选最佳质量、最适用量和浓度的吐温80,既能保证助溶效果,又能最大限度的提高制剂安全性和制剂稳定性。4羟丙基-β-环糊精助溶效果好、稳定,无明显毒性、致敏性及溶血性,是目前比较理想的增溶剂。以羟丙基-β-环糊精替换掉吐温80的鱼腥草注射液新制剂,其溶解性、安全性和药效不受影响,为其恢复生产和临床应用提供了科研依据。5诱发鱼腥草注射剂临床严重不良反应因素除了药物自身因素,制剂工艺中加入吐温80以及临床不合理用药外,还存在很多偶发因素。因此,本研究只解决了中药注射剂安全性事件中的一个因素,而不是唯一因素,还需要深入开展多方面研究,为中药注射剂进行全面再评价和再研究提供思路和方法。本研究的创新点如下：1比较中药注射剂导致类过敏和过敏反应的异同,主要从临床反应、病机、诱发物质、给药次数、剂量、检测指标、非临床评价方法和配伍禁忌进行了概括和总结；2采用不同种属动物进行类过敏实验研究,优选实验动物、检测指标,优化致敏性评价方法,建立中药注射剂类过敏反应的评价方法和指标；3通过一系列药学、安全性和比较药理学研究,证实以羟丙基-β-环糊精为增溶剂的鱼腥草注射液新制剂的安全性显著提高,药效与原制剂相当,是比较有开发和应用前景的鱼腥草注射剂。更多还原

【Abstract】 The re-evaluation and re-research of Houttuynia Cordata injection, as the CTMI clinical adverse reactions occurred representatives, are very practical significance. The safety re-evaluation of Houttuynia Cordata injection was be carried out on comprehensive scientific assessments, such as:preparation process, quality standards, pharmacology and toxicology, clinical use and drugs economics. Systematic analysis of the clinical adverse reaction causesd by Houttuynia injection, clear the main allergens, improve production quality standards and preparation process, inhance Houttuynia injection safety, effectiveness and stability, in order to restore production and provide scientific basis for clinical applications.1, Literature ReviewIn the part of literature review, we discussed three aspects:①The reseach of Houttuynia and its preparations;②The analysis and strategy of clinical adverse reaction by Houttuynia injection;③Research for injection accessories of Tween 80 and hydroxypropyl-β-Cyclodextrin. These put forward the theoretical basis for the overall study.2, Study on evaluation of allergenicity of original, new Houttuynia injection and different auxiliariesThis study on allergenicity evaluation the original, new Houttuynia injection and different auxiliaries for used mice, Beagle dogs and rhesus macaque. Anaphylactoid experiment, with single intravenous dose of constant injection, observed changes in behavior of animals after administration and the concentration of histamine. Allergy test was carried out, through intravenous injection of three times to Beagle dogs to immunize, and inject double doses of injections to stimulate in the 9 days after the last sensitized. Observe the allergy situation of Beagle dogs and measure the concentrationns of histamine, IgE in plasma with ELISA after administration in order to determine results. The results show that Beagle dogs to anaphylactoid reactions is the best experimental animal, behavior disorders and histamine concentration as the main index, IgE as the secondary indicator. Rhesus macaque under the same conditions is not sensitive. Atypical behavior and blood indexes in mice are not occurred, its results are not for reference. Tween 80 is the representation of substance-induced anaphylactoid reactions. The results of Hydroxypropyl-β-cyclodextrin in anaphylactoid and allergic reactions are negative. The results in allergenicity evaluation from new Houttuynia cordata injection with hydroxypropyl-β-cyclodextrin as solubilizer are also negative.3, Study on comparative pharmacology of new, original Houttuynia intravenous injectionsIn this study, the model of infected mice with pneumonia influenza virus PR8 and FM1 strains in vivo, CPE in vitro and neuraminidase Fluorescence, viral load testing and death to protect experimental methods were used.The results show that the new Houttuynia injection had obvious effects of treatment and prevention for the pulmonary inflammation resuting form influenza virus infection in mice, improving the pneumonia symptoms, reducing the lung index and viral load, reducing mortality and prolong survival time; but its antiviral activity is not inhibited or killed by the virus directly, the neuraminidase activity is not inhibited. The new preparation could significantly inhibit the swelling degree of mice ear induced by dimethyl benzene and the effusion of Evans blue from capillary permeability induced by acetic acid in mice; they also could decrease the rectal temperature induced by yeast liquid in rats and LPS-induced in rabbit fever. Anti-inflammatory and antipyretic of Houttuynia injection may be inhibitied by the inflammatory cytokines TNF-α, IL-1β, IL-8 and PGE2. Anti virus, anti-inflammatory and antipyretic of new, original Houttuynia injection are quite.4, Establishment of qualitative and quantitative determination of Tween 80 in Houttuynia injectionAmmonium cobalt thiocyanate color method, thin layer chromatography and bismuth potassium iodide was used to the qualitative identification of Tween 80, the results are consistent. Quantitative analysis of Tween 80 can accurately detect the concentration of Tween 80 in TCMI. Qualitative and quantitative methods of Tween 80 can at randomly detect the samples of different manufacturers and batches of TCMI containing Tween 80 under laboratory conditions.The conclusions1, Comparison the similarities and differences between allergy and anaphylactoid reactions induced by TCMITCMI lead to serious adverse in clinical over 70%of anaphylactoid reactions, different to classical IgE-mediated antibody-antigen response. Main features of anaphylactoid reactions are:First injection, serious adverse reactions can be arised, such as anaphylactic shock, and similar syndrome with allergic reactions. It is non-antigen-antibody reaction, no significant changes in IgE and IgG, but histamine significantly increased in vivo. The allergenic substance is antigen or hapten as well as histamine-induced release substances. Obvious dose-effect relationship, it can induce serious adverse in high dose and concentrations. Combined with a variety of injections can increase the incidence and severity. Beagle dogs to anaphylactoid reactions is the best experimental animal, behavior disorders and histamine content as the main index, IgE as the secondary indicator. Therefore, assessment of sensitization in Beagle dogs should be a routine safety testing methods of TCMI in Pharmaceutical factory.2, The major allergenic substances in Houttuynia cordata injection is Tween 80, Houttuynia double-distilled liquid is no obvious side effects.The allergens triggerred adverse reactions of TCMI should account into the drug itself factors, plused accessories in preparation as well as the exogenous and endogenous contaminants in the torage process, which provide ideas in the identification of adverse drug reactions induced by TCMI.3, Tween 80 are widely used at home and abroad. Tween 80 in 134 varieties of TCMI added uncertain.If Tween 80 of all of these species will be replaced must affect the survival of hundreds of manufacturers and hundreds of thousands of industrial workers, social stability and economic impact must be carefully taken into account.4, Hydroxypropyl-β-cyclodextrin is the ideal solubilizer, which has the good solubilization effect, stability, and no significant toxicity, sensitization, hemolytic. New Houttuynia injection with Hydroxypropyl-β-cyclodextrin as solubilizer has good solubility, safety and efficacy, these provide scientific basis for restoring production and clinical applications.5, The factors of Houttuynia injection induced clinical serious adverse are not self-medication, adding Tween 80 in preparation process as well as clinical irrational drug use, but also many incidental one.This study only solve one of the factors of the safety incident of TCMI, but not the all, many studies also need in depth, which will put forward the further research ideas and methods for the comprehensive re-evaluation of CTMI.Innovation of this study1, Comparison with the differences in anaphylactoid and allergic reactions lead to by TCMI:clinical syndrome, pathogenesis, inducing substances, frequency of administration, dosage, test indicators, non-clinical evaluation methods and incompatibility;2, Using different animal experiments in the anaphylactoid research, purpose is the optimization of experimental animals, detection index and allergenicity evaluation method, which can establish the evaluation methods and indicators of anaphylactoid reactions by TCMI;3, Through a series of pharmacy, security research and comparative pharmacology, The new Houttuynia cordata injection with hydroxypropyl-β-cyclodextrin as solubilizer significantly improves the safety, the same efficacy with the original preparation, it has better prospects of development and application in the future.更多还原